1. Field of the Invention
The invention relates to peroxidase colloidal gold biosensors that provide a detectable electrochemical response based on direct oxidation of a redox protein at an electrode surface. In particular, mediatorless detection of glucose is possible with colloidal gold adsorbed horseradish peroxidase in the presence of glucose oxidase. The invention also includes methods of mediatorless detection of various analytes and processes for the preparation of colloidal gold adsorbed peroxidase based bioelectrodes.
2. Description of Related Art
Direct electron transfer between an enzyme and an electrode surface is of practical as well as theoretical interest. An enzyme capable of direct electron transfer immobilized on an electrode permits electrochemical measurement of the enzyme substrate without addition of a mediator to the analyte solution. Unfortunately, a serious problem with protein electrochemistry is the slow mass transport process and strong adsorption of protein molecules onto the electrode surface.
Because of the tendency of protein molecules to adsorb to surfaces, direct electron transfer to or from the electrode surface is possible only for the first layer of protein on the electrode. Even assuming a monolayer coverage and completely reversible electrochemistry between the adsorbed monolayer and the electrode surface, direct electron transfer between an adsorbed monolayer of redox protein and an electrode surface would result in a current approximately one-half that of the charging current.
While there are some examples of detectable electrochemical response based on direct oxidation of a redox protein at an electrode surface, detection has been difficult (Joensson and Gorton, 1989; Bowden et al., 1984). Amplification of the signal can in some cases be achieved by adding enzyme substrate.
Generally, in order to detect a signal, substrate is added in order to induce enzyme turnover (Guo and Hill, 1991). This significantly amplifies the signal which otherwise is generally too weak to be detected. A few limited examples showing direct electron transfer between various enzymes and electrode surfaces include cytochrome c peroxidase (Armstrong and Lannon, 1987), p-cresolmethylhydroxylase (Gou and Hill, 1989), and cytochrome c.sub.552 (Guo and Hill, 1990) at surface-modified electrodes or in the presence of promoters. Other examples include cytochrome c peroxidase irreversibly adsorbed on pyrolytic graphite (Paddock and Bowden, 1989), and lysyl oxidase (Govindaraju et al., 1987) and horseradish peroxidase (Joensson and Gorton, 1989) on spectrographic graphite.
Current theories of non-mediated electrochemistry of proteins and enzymes emphasize the importance of the electrode surface in facilitating direct electron transfer (Guo and Hill, 1991). It has also been suggested that direct electron transfer may proceed most easily to/from electrode surfaces which provide an environment similar to the native environment of the redox protein (Armstrong, 1991). However, there has been limited success with approaches that deposit the redox protein directly on the surface, presumably because of protein denaturation.
Horseradish peroxidase (HRP) has been suggested and studied as a bioelectrode. An HRP electrode has high specific activity for H.sub.2 O.sub.2 with each H.sub.2 O molecule effectively converting ca. 25,000 H.sub.2 O.sub.2 molecules to H.sub.2 O per minute. In the presence of H.sub.2 O.sub.2, HRP is efficiently converted to its oxidized form, HRP.sub.ox (reaction (1)) (Frew et al., 1986). This can then be reduced, as shown in reaction (2), either directly or through an electron transfer mediator acting as an electron shuttle (Frew et al., 1986). EQU H.sub.2 O.sub.2 +HRP.sub.red .fwdarw.HRP.sub.ox +H.sub.2 O (1) EQU HRP.sub.ox +2e.sup.- .fwdarw.HRP.sub.red ( 2)
While electrodes based on horseradish peroxidase Will demonstrate direct electron transfer (Joensson and Gorton, 1989), a major problem in developing a redox system utilizing HRP has been to induce the heterogeneous electron transfer step (reaction step 2) to proceed at a reasonable rate. Acceptable rates of transfer are obtained in the presence of a mediator, but without a mediator the rates are too slow to be of practical value.
Biosensors are of particular interest for measuring glucose and there are biosensors utilizing glucose oxidase as the sensing enzyme. A glucose sensor based on gel immobilized glucose oxidase detects changes in pH when coimmobilized with gluconolactase which hydrolyzes the lactone product of glucose oxidation (Nakamoto, 1992). This type of glucose is, however, relatively insensitive to glucose levels below about 0.1 mM.
More sensitive enzyme electrochemical sensor electrodes have been developed that employ polymeric surface coatings. An enzyme such as glucose oxidase dispersed in the polymer facilitates detection of hydrogen peroxide produced during the reaction when employing a system incorporating a reference/counter electrode with the enzyme-coated electrode (Rishpon et al., 1992).
As a general principle, in the operation of a glucose biosensor, glucose oxidase is reduced during the oxidation of glucose; the reduced enzyme is then reoxidized either through an electron transfer mediator, which itself becomes reoxidized on the electrode surface, or through molecular oxygen present in the solution. The product resulting from oxygen reduction is hydrogen peroxide which can be reoxidized at the electrode at high positive potential, or, reduced to water at a high negative potential. In either case, a high background signal is generated with high risk of interferences from the sample matrix.
On the chemical level, a glucose biosensor is based on the conversion of glucose (GO, the substrate or analyte) to gluconolactone (GL) in the presence of a catalyst, glucose oxidase (GOD), represented by the following equation: EQU GO+GOD.fwdarw.GL+GOD.sub.red ( 3)
In order to maintain continuous oxidation of GO, GOD.sub.red has to be reoxidized to GOD. Equations 4-6 represent three different paths for recycling GOD. ##EQU1## The added electron transfer agent or mediator may be reoxidized as shown in equation (7) EQU MED.sub.red -e.sup.- .fwdarw.MED.sub.ox ( 7)
Hydrogen peroxide generated from reduction of molecular oxygen will react, depending on conditions, in the reduction mode, equation (8), or in the oxidation mode, equation (9). EQU H.sub.2 O.sub.2 +2 e.sup.- +2 H.sup. .fwdarw.2 H.sub.2 O (8) reduction mode EQU H.sub.2 O.sub.2 -2 e.sup.- .fwdarw.O.sub.2 +2 H.sup.+ ( 9) oxidation mode
The process represented by equation (4) is normally very slow and therefore considered impractical. The reaction with molecular oxygen, equation (5), will take place unless oxygen is purged from the system. Mediated reactions, represented by equation (6), can be quite efficient, depending on the mediator.
For purposes of developing a practical glucose biosensor, three options would include, based on equations 3-9:
Mode one: (3).fwdarw.(6).fwdarw.(7): oxidation mode; PA1 Mode two: (3).fwdarw.(5).fwdarw.(9): oxidation mode; and PA1 Mode three: (3).fwdarw.(5).fwdarw.(8): reduction mode
Mode one operates at a potential of 0.3-0.4 V and has the advantage of being a direct measure of the glucose oxidase redox process. There are, however, several disadvantages, including requirement of a mediator which to be effective must be immobilized near the electrode surface. The effectiveness, operational potential (0.3-0.4V/Ag/AgCl) and the background current depend on the mediator. Moreover, the mediator must be initially in its oxidized form in order to minimize the initial background current. Unfortunately, good mediators, e.g., ferrocene and its derivatives, are only readily available in their reduced form.
Yet another disadvantage of Mode one operation is sensitivity to molecular oxygen. O.sub.2, when present, will compete with the mediator. As a practical matter, purging the oxygen is time-consuming and expensive in large scale operations. The effect of O.sub.2 depends on the relative rate of the reactions shown in equations (5) and (6). A further disadvantage is the dependence of the O.sub.2 effect on glucose concentration as well as the concentration of molecular oxygen present. Variation of ambient O.sub.2 concentration therefore will have unpredictable effects on the mediated signal. Even at constant O.sub.2 concentration, predictability is difficult because the effect is more detrimental at low glucose concentrations than at higher glucose concentrations (Hale et al., 1991; Gregg and Heller, 1990). At present, no mediators have been reported that operate efficiently enough to eliminate the oxygen effect.
Mode two operates at a potential of 0.6-0.7V and has several advantages, including the fact it is not sensitive to oxygen at low glucose concentrations as there is usually sufficient oxygen in the solution. Additionally, a mediator is not required and there are no competitive reactions, assuming no interfering substances are added in the sample.
Mode two does, however, have several disadvantages. The process is sensitive to oxygen at high glucose concentrations when oxygen which is normally present may become limited. The product, not the enzyme redox process, is measured. And the high operational potential, 0.6-0.7 V/Ag/AgCl, results in a high background current, so that the signal current may be difficult to detect.
Mode three operates at 0V Ag/AgCl and has a number of advantages. This system can be coupled to HRP with direct electron transfer in the reduction mode, equation (8), at 0V on the electrode. As in Mode two, no mediator is required, there are no competing reactions and there is no oxygen sensitivity at low glucose concentrations. A distinct advantage is low background and interference due to the low operational potential.
Mode three disadvantages include sensitivity to oxygen at high glucose concentrations and measurement of a product rather than the enzyme redox process directly. Additionally, two enzymes are required, adding complexity to the system and possible additional expense for fabrication.
Enzyme electrochemical sensors for glucose determination have been described (Rishpon et al., 1992). In these Mode one type biosensors, GOD is incorporated into membranes near the electrode surface to reduce interference from undesired oxidizable compounds and to reduce oxygen sensitivity. The electrode is however not sensitive to glucose concentrations below about 1 mM.
Electron transfer agents, such as ferrocenes, have been used in conjunction with glucose oxidase. However, two major drawbacks exist. In common practice, electron transfer mediators are small molecules, typically ferrocene for glucose oxidase based biosensors. It is generally desirable to immobilize a mediator to keep it close to the surface; however, small molecules are difficult to immobilize. A more difficult problem is the ubiquitous presence of molecular oxygen. Oxygen will always be reduced to some extent, even in the presence of a mediator. The result is that, while a mediated response may produce a satisfactory response to relatively high glucose concentrations, it is not feasible to measure low glucose (100 .mu.M range) concentrations because of background current and the effect of oxygen.